The regulation of gene expression requires the coordinated action of proteins with regulatory and/or enzymatic activities. Many of the enzymes catalyze the posttranslational modifications of the histone proteins within chromatin to either facilitate gene activation or repression/silencing. One such family of enzymes is the histone acetyltransferases (HATs) that acetylate the epsilon amino group of specific lysine residues within the N-terminal tail regions of histones, as well as some non-histone proteins, to promote gene activation. In vivo, HAT enzymes function in the context of multisubunit complexes and fall into subfamilies containing sequence conservation and related substrate specificities. Over the last funding period, we have used X-ray crystallography and complementary biochemical and enzymatic analysis to characterize the histone substrate binding and catalytic properties of the Gcn5/PCAF and MYST HAT families. As a model to understand how different histone modifications can work synergistically for gene activation, we also characterized the mechanism by which phosphorylation by the Snfl histone kinase facilitates acetylation by Gcn5 at histone H3. Finally, we have provided the first structural scaffold for a HAT/inhibitor complex. Together, our studies have provided a paradigm for understanding the mechanism for post-translational histone modification by HAT enzymes. The overall objective of the current proposal is to further our understanding of catalysis and substrate binding specificity by HAT proteins of the Gcn5/PCAF and MYST families, as well as of the global transcriptional regulator HAT, p300/CBP. We will also extend our studies as to how histone acetylation is modulated by other histone modifications and by other protein subunits within in vivo HAT complexes. We will continue to use a combined approach of high resolution X-ray structure determination and functional characterization in vitro. The specific aims of the proposal are to (1) Characterize the structure and binding properties of the Gcn5/PCAF HATs bound to relevant histone and non-histone substrates and bound to bisubstrate inhibitors; (2) Determine structures of the MYST family of HAT proteins in complex with histone peptide substrates; (3) Characterize the structure/function of the human p300/CBP HAT protein; (4) Characterize the structure/function of the multisubunit Sas2/4/5 HAT complex, and (5) Characterize the structure/function of the Snfl histone H3 kinase. These studies will provide new molecular insights into the substrate specificity and catalytic mechanism of the Gcn5/PCAF, MYST and p300/CBP family of histone acetyltransferases, and how these enzymes may coordinate with other regulatory proteins and other histone modifications to modulate gene expression. Moreover, these studies will provide a scaffold for the design of small molecule inhibitors for specific histone acetylase enzymes that may have applications in basic research and medicine.